This invention relates to synthesis of nucleic acids, and more particularly to synthesis of nucleic acids by a polymerase chain reaction method.
The polymerase chain reaction (hereafter PCR) method is one of the techniques for amplifying genes. By this method, a particular segment of target DNA (template DNA) can be amplified by a factor of hundreds of thousands, and this is accomplished with a pair of primers sandwiching target DNA regions and by repeating reactions consisting of denaturation, annealing and polymerization of DNA. The PCR is usually used for the analysis of an extremely small amount of DNA, for example, from one virus, one bacterium or one cell in samples under investigation.
For the analysis of nucleic acids of viruses, bacteria or cells contained in samples from living organisms (such as tissue, blood, marrow fluid, sputum, milk, urine and feces of an animal or roots, stems, leaves, flowers and fruits of a plant), an environmental sample (such as soil and water) or a food sample (such as meats, milk and eggs), however, it is necessary to first remove impurities in the sample and to separate and purify the nucleic acids. A commonly practiced method for this process at the present time is to first treat the sample with an enzyme or a surfactant and then to use a phenol-chloroform mixture. More recent methods of purifying DNA include the removal of impurities by using ion exchange resins, glass beads and agents for protein separation. By any of these methods, however, it is difficult to sufficiently remove the impurities, and this frequently causes to inhibit the amplification of genes by a method of synthesizing nucleic acids such as the PCR method.
Hypothesizing that the addition of polyamine during gene amplification might reduce the inhibition of the synthesis of nucleic acids by impurities, the present inventors extensively reviewed available documents and reports. Wan and Wilkins (PCR Methods and Applications, 3, 208-210 (1993)) reported that spermidine, which is a kind of polyamines, facilitates PCR amplification of high-quality genomic DNA purified from cotton leaves by tedious purification procedures and that optimum spermidine concentrations are 0.2-1 mM, but there has not been any report describing that the addition of polyamine could reduce the inhibition of the synthesis of nucleic acids by impurities contained in samples or low-quality DNA purified from samples by rough purification procedures.
It is therefore an object of this invention to provide methods of synthesizing nucleic acids from a sample which is likely to inhibit synthesis of nucleic acids, that is, a crude sample obtained from materials derived from living organisms.
For the purpose of attaining the aforementioned object, the present inventors investigated whether polyamine reduced the inhibition of nucleic acid synthesis by the existence of impurities by adding various kinds of polyamine at various concentrations to crude samples from materials such as human feces and blood, and thereafter using a PCR method to carry out the synthesis of nucleic acids. As a result, it was discovered that the inhibition of nucleic acid synthesis by impurities in the sample can be reduced if any of various kinds of polyamine is added at a concentration within a certain range.